title: recycling old drugs: cardiac glycosides as ... · 15/04/2020 · identifying cardiac...

Title: Recycling old drugs: cardiac glycosides as therapeutics to target barrier 1 inflammation of the vasculature, meninges and choroid plexus 2

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One Sentence Summary 4

We have identified cardiac glycosides as powerful regulators of neuroinflammatory 5 pathways in brain-barrier tissues such as vasculature, meninges and choroid plexus. 6

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Authors: Deidre Jansson1,3,11 , Victor Birger Dieriks2, Justin Rustenhoven9,10, Leon C.D. 8 Smyth4,5, Emma Scotter1, Miranda Aalderink1,3, Sheryl Feng1,3, Rebecca Johnson1,3, Patrick 9 Schweder7, Edward Mee7, Peter Heppner8, Clinton Turner6, Maurice Curtis2, Richard Faull2, 10 and Mike Dragunow1,3* 11

Affiliations 12 1Department of Pharmacology and Clinical Pharmacology, The University of Auckland, 13 Auckland, New Zealand. 14 2Department of Anatomy and Medical Imaging, The University of Auckland, Auckland, New 15 Zealand. 16 3Hugh Green Biobank, Centre for Brain Research, The University of Auckland, Auckland, New 17 Zealand. 18 4Department of Pathology, University of Otago, New Zealand. 19 5Centre for Free Radical Research, University of Otago, Christchurch, New Zealand 20 6Department of Anatomical Pathology, LabPlus, Auckland City Hospital, Auckland New 21 Zealand 22 7Department of Neurosurgery, Auckland City Hospital, New Zealand. 23 8Starship Hospital, Auckland, New Zealand 24 9Center for Brain Immunology and Glia (BIG), University of Virginia, Charlottesville, VA, USA . 25 10Department of Neuroscience, School of Medicine, University of Virginia, Charlottesville, VA, 26 USA. 27 11Department of Psychiatry and Behavioural Science, University of Washington, Seattle, WA, 28 USA 29

30 *Corresponding author: Professor Mike Dragunow. 31 Address: Department of Pharmacology and Clinical Pharmacology, The University of 32

Auckland, Private Bag 92019, 1142, Auckland, New Zealand. 33 Telephone: 0064-9-923 6403 34 Fax: 0064-9-373 7556 35 Email: [email protected] 36 37

Abstract 38 39 Neuroinflammation is a key component of virtually all neurodegenerative diseases; 40 preceding neuronal loss and associating directly with cognitive impairment. 41 Neuroinflammatory signals can originate and be amplified at barrier tissues such as brain 42 vasculature, surrounding meninges and the choroid plexus. We designed a high-throughput 43

(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted April 17, 2020. ; https://doi.org/10.1101/2020.04.15.043588doi: bioRxiv preprint

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https://doi.org/10.1101/2020.04.15.043588

screening system to target inflammation in cells of the blood-brain barrier (primary human 44 pericytes and endothelia) and microglia enabling us to target human disease-specific 45 inflammatory modifiers. Screening an FDA-approved drug library we identified digoxin and 46 lanatoside C, members of the cardiac glycoside family as inflammatory modulating drugs that 47 work in blood-brain barrier cells. A novel ex vivo assay of leptomeningeal and choroid plexus 48 explants further confirmed that these drugs maintain their function in 3D cultures of brain 49 border tissues. While current therapeutic strategies for the treatment of neurodegenerative 50 diseases are missing the mark in terms of targets, efficacy and translatability, our innovative 51 approach using in vitro and ex vivo human barrier cells and tissues to target 52 neuroinflammatory pathways is a step forward in drug development and testing, and brings 53 us closer to translatable treatments for human neurodegenerative disease. 54 55

Key Words 56

Digoxin, Lanatoside C, pericytes, endothelia, blood-brain barrier, inflammation, high 57 throughput screening, human primary brain cells 58

Background 59

Dysregulated chronic inflammatory responses occur early in disease progression, contribute 60 to blood-brain barrier dysfunction and can precipitate neurodegeneration (1-5). 61 Accumulating evidence suggests an inflammatory contribution by cells of the 62 neurovasculature, meningeal compartments and choroid plexus (CP) by facilitating the 63 communication of inflammatory signals from the periphery and within the CNS (6, 7). Since 64 neuroinflammation in the central nervous system (CNS) can precede and exacerbate neuronal 65 loss, attenuation of these responses in contributing cell types prior to overt deterioration 66 should be more appropriately explored as a therapeutic option (8-10). 67

Neuroinflammation can be most easily targeted anatomically at several sites; the 68 cerebrovasculature that reaches the entirety of the CNS bordered luminally by the blood, and 69 abluminally by the cerebrospinal fluid (CSF), the meninges which surround and envelop the 70 brain and spinal cord directly communicating with lymphatic vessels, and the CP that 71 produces CSF and facilitates fluid movement by way of glymphatic flux (11-13). It has been 72 suggested that the brain’s immune functions have been concentrated at these border tissues, 73 which may be more sensitive to inflammatory cues than the rest of the CNS (14-16). We have 74 designed our study to test inflammatory modulating drugs on the spectrum of cell types that 75 make up these structures. 76

At the cellular level the neurovascular unit (NVU) is formed by the specialized arrangement 77 of endothelia, pericytes, astrocyte endfeet, and neurons that work together to maintain brain 78 homeostasis. We have demonstrated that pericytes derived from post-mortem human brains 79 display a robust inflammatory response when stimulated by classical inflammatory mediators 80 such as tumor necrosis factor α, interferon γ (IFNγ), interleukin 1β (IL-1β) and 81 lipopolysaccharide (LPS), and are likely to be targets of neuroinflammation in both an 82 autocrine and paracrine fashion (17, 18). Importantly, endothelia and pericytes in the NVU 83 contribute to neuroinflammatory responses by chemokine secretion, adhesion molecule-84 mediated leucocyte extravasation, and damage-associated molecular patterns (DAMPS) and 85 pathogen associated molecular patterns (PAMPS) receptor expression (11, 19-22). 86


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Attenuating the infiltration of peripheral immune cells into the brain or preventing 87 inflammatory propagation by barrier cells such as neurovascular cells is likely to be beneficial 88 in limiting CNS immune responses, although few CNS therapies have been based upon this 89 approach. Namely the MS drug natalizumab that targets immune cell entry into the CNS is a 90 good example of this approach (23). In this study in addition to primary human brain 91 neurovascular cells such as pericytes, endothelia cells and microglia, we also made use of 92 explants from barrier tissues such as meninges and CP which are directly involved in 93 inflammation. 94

The human meninges are composed of three membranes (dura, arachnoid, and pia mater) 95 that envelop the brain and spinal cord. The arachnoid and pia mater, collectively termed the 96 leptomeninges, form a semi-permeable membrane to the CSF which fills the subarachnoid 97 space. The leptomeninges are comprised of several cell types, including macrophages, 98 dendritic cells, mast cells, and fibroblasts and are permeated by leptomeningeal arteries (24, 99 25). Furthermore, diverse leucocyte populations reside in the subarachnoid space providing 100 a conduit for immune-brain communication (26-28). Meningeal inflammation often precedes 101 inflammation in the CNS and is present in neurodegenerative diseases, chronic inflammatory 102 conditions, and acute pathogen introduction (29-31). Importantly, meningeal-derived factors 103 can permeate the brain parenchyma, presumably through glymphatic influx, suggesting that 104 modulating meningeal-derived inflammation also represents an appropriate target to prevent 105 inflammatory-mediated CNS insults (32, 33). 106

The CP is a highly vascularised tissue that harbours fenestrated capillaries surrounded by 107 apical facing, CSF-producing epithelial cells and serves as a gateway for immune cells from 108 the circulatory system to the CNS (34). The fact that the CP is a sink for immune cells is of 109 particular importance when it comes to immune cell trafficking in both sterile inflammatory 110 and chronic inflammatory conditions (35, 36). Similarly, the CP itself increases secretion of 111 inflammatory messenger molecules into the CSF with age and disease, which are transported 112 throughout the brain through CSF circulation (37, 38). Together this supports the examination 113 of the CP as a target for reducing neuroinflammation. 114

Here, we utilise primary dissociated human brain pericytes and endothelia, as well as explants 115 from the meninges and the CP to screen drugs for anti-inflammatory properties. Drug 116 screening approaches are not typically performed using primary human brain cells due to 117 limited tissue yields, difficulties of human brain cell culture and accessibility. However, rodent 118 immune cells display numerous discrepancies compared to their human counterparts, with 119 respect to both immune functions and pharmacological responses (32, 39), necessitating the 120 use of primary human brain cells for pre-clinical compound screening with an intent for 121 translational drug discovery. Primary human brain pericytes are suitable for high throughput 122 drug screening for candidate compounds with anti-inflammatory functions because unlike 123 many other human brain cell types, pericytes undergo rapid proliferation in vitro, allowing for 124 efficient bulking of these cells for the purposes of high-throughput drug screening. In this 125 study, selected hits from initial screens were validated in both pericyte and endothelial 126 cultures to explore the anti-inflammatory efficacy of these compounds in NVU-associated 127 cells. Next we describe and characterise the inflammatory contribution of an ex vivo model of 128 human leptomeningeal and CP explants using it to investigate lead anti-inflammatory 129 compounds in a complex multicellular system more closely recapitulating the in vivo human 130 brain environment. Finally, we demonstrate the efficacy of two anti-inflammatory 131


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compounds digoxin and lanatoside C, in attenuating meningeal inflammatory responses and 132 preventing meningeal-mediated inflammatory propagation. 133

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Results 135

Identifying cardiac glycosides in a screen using primary pericytes 136

Primary human brain pericytes respond to inflammatory stimuli to induce the expression of 137 chemokines and adhesion molecules, including chemokine (C-C motif) ligand 2 (CCL2) and 138 intercellular adhesion molecule-1 (ICAM-1), respectively (17, 40-42). Due to their involvement 139 in enhancing leucocyte infiltration into the brain, these mediators were selected as candidate 140 proteins to determine the anti-inflammatory potential of the Prestwick compound library 141 (17). Concentration response curves of IL-1β-induced CCL2 and ICAM-1 protein expression, 142 as determined by immunocytochemistry, revealed an EC50 of 0.02 ng/mL for CCL2 and 0.03 143 ng/mL for ICAM-1, thus 0.05 ng/mL was selected as an optimal concentration of IL-1β to 144 determine immune modulation, allowing for identification of compounds which may induce 145 and attenuate inflammatory responses (Fig. 1A and B). Pericytes were screened for 146 modulators of IL-1β-induced CCL2 and ICAM-1 expression by pre-treating cells with > 1200 147 FDA approved compounds at 10 µM in duplicate for 24 hours, then stimulating with IL-1β 148 (0.05 ng/mL) for 24 hours. CCL2 and ICAM-1 intensity values normalized to total cell counts 149 confirmed inhibitory effects of transforming growth factor receptor β (TGFβ1), a known 150 inhibitor of chemokine and adhesion molecule expression in pericytes (42) (Fig. 1C and D). 151 Whilst some plate variability in terms of cell numbers and absolute intensities was observed, 152 the IL-1β induction of CCL2 or ICAM-1, and the TGFβ1-dependent inhibition was consistent 153 across all test plates (fig. S1). A total of 82 compounds that met cut-off criteria (changes in 154 intensity ± 20 % over vehicle + IL-1β values, < 50 % cell loss, with standard deviations of < 15 155 % for either CCL2, ICAM-1, or both) were selected for further validation. Drug classification 156 revealed hit compounds that altered CCL2 or ICAM-1 were mainly comprised of therapeutics 157 categorised as anti-bacterial, anti-asthmatic, anti-helminthic, cardiotonic, bronchodilator, 158 anti-septic, anti-neoplastic, anti-amoebic, and anti-inflammatory (Fig. 1E and F). A second 159 confirmatory screen of the 82 hits generated 44 that had reproducible effects. These 44 hits 160 were then screened at three concentrations (0.1, 1, and 10 µM) for the ability to modify IL-161 1β-induced CCL2 or ICAM-1 in pericytes, without causing significant cell loss (Trial 3, fig S1). 162 IL-1β-independent effects were assessed by screening compounds in pericytes with the same 163 paradigm (24 hours of compounds at 10 µM, 24 hours of vehicle only for IL-1β), which 164 revealed no significant changes relating to CCL2 or ICAM-1 expression (fig. S1). 165

Hits were narrowed down to 10 compounds that consistently modified either IL-1β-induced 166 CCL2 or ICAM-1 expression by immunocytochemistry without significant cell reduction (as 167 assessed by total cell counts). To further investigate the anti-inflammatory ability of hit 168 compounds, their effect on secretions of a larger panel of inflammatory chemokines, 169 cytokines, and adhesion molecules previously identified to be secreted by pericytes in 170 response to IL-1β was determined by cytometric bead array (CBA) (43). Concentrations 171 determined from Trial 3 were used to treat pericytes as above (fig. S1). Analysis of cytokine 172 secretion showed that both cardiac glycosides digoxin and lanatoside C had the most 173 substantial effects on soluble ICAM-1 (sICAM-1) and fractalkine (CX3CL1) (a known microglial 174 ligand) (44). In particular, digoxin inhibited IL-1β-induced secretion of CCL2, sICAM-1, soluble 175


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vascular cell adhesion molecule-1 (sVCAM-1), and CX3CL1 but increased secretion of 176 interleukin-6 (IL-6) more than 6-fold over IL-1β alone (Fig. 1G, fig S2). A generalized pipeline 177 for refinement of the hit compound list leading to digoxin as a lead compound is presented in 178 Fig. 1H. Digoxin is indicated for the treatment of symptomatic heart failure, and atrial 179 fibrillation, thus we were surprised to see anti-inflammatory effects on vascular cells. 180 Lanatoside C, another cardiac glycoside found in the Prestwick library, demonstrates 181 structural similarity with digoxin (45). Little is known concerning inflammatory outputs by 182 cardiac glycosides on brain vascular cells, and due to the ability of digoxin to most effectively 183 limit pericyte-mediated inflammatory responses, digoxin and the closely-related lanatoside C 184 where chosen as lead compounds for further analysis. 185

Anti-inflammatory effects of digoxin and lanatoside C in primary human brain pericytes 186

Pericytes express several proteins that facilitate leukocyte extravasation and polarise 187 surrounding immunologically-active cells to pro- or anti-inflammatory phenotypes (41, 46). 188 Secretion data from pericytes pre-treated with digoxin and lanatoside C were consistent with 189 an inhibitory effect on IL-1β-induced soluble adhesion molecule (sICAM-1 and sVCAM-1) and 190 CX3CL1 secretion, whilst IL-6 was increased and interkeukin-8 (IL-8) displayed no change (Fig 191 2 A-L, concentration response curves (fig. S2A)). Because complications often arise with 192 excessive dosage of digoxin, several outputs of pericyte viability following digoxin or 193 lanatoside C treatment were examined to rule out the possibility of the inflammatory-194 modulating effects being a result of cytotoxicity. While both digoxin and lanatoside C reduced 195 pericyte number, this is likely a reflection of reduced proliferation over the treatment period 196 (fig. S2B), as opposed to cell death, which was largely unchanged. Interestingly, a reduction 197 in PDGFRβ expression was observed in response to digoxin and lanatoside C that was 198 consistent with reduced proliferation at this time point. Signalling through the platelet-199 derived growth factor receptor β (PDGFRβ) pathway in pericytes promotes their proliferation 200 (47, 48) suggesting that the anti-proliferative effect of digoxin/lanatoside C may result from 201 this PDGFRβ depletion. 202

Transcription factor activation is a key component of the inflammatory response, in both 203 negative and positive regulatory pathways. CCAAT/enhancer binding protein delta (CEBPδ) 204 has recently been identified as an anti-inflammatory effector in pericytes, downregulating IL-205 1β-induced CCL2 and ICAM-1 expression (43). Conversely, in response to IL-1β, TNFα, and LPS, 206 nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) translocates to the 207 nucleus where it induces transcription of numerous pro-inflammatory transcripts (17, 49). 208 Lanatoside C induced CEBPδ nuclear expression under basal conditions, while both digoxin 209 and lanatoside C increased IL-1β -induced expression (Fig. 2M and N). In contrast both 210 compounds reduced IL-1β-induced NFκB nuclear translocation (Fig. 2O and P) and TGFβ1-211 induced SMAD2/3 translocation (fig. S3A and B), with no effect on IFNγ-induced signal 212 transducer and activator of transcription 1 (STAT1) activation (fig. S3C and D), suggesting a 213 selective element of inflammatory pathway inhibition. Further inspection of NFκB pathway 214 components from pericyte cell lysates revealed reduced activity of known upstream elements 215 including myeloid differentiation primary response 88 (MyD88), interleukin-1 receptor (IL-1R) 216 associated kinase (IRAK), nuclear factor of kappa light polypeptide gene enhancer in B-cells 217 inhibitor, alpha and epsilon (IκBα, IκBε) and TNF receptor signalling, at the same time 218 specifically increasing phosphorylation of NFκB/p65 at S529 (Fig. 2 Q and R). Interestingly, 219 gene expression analysis of pericytes under the above conditions do not show a diminished 220


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inflammatory response- quite the opposite. Both digoxin and lanatoside C increased 221 transcripts for CCL2, ICAM-1, VCAM-1, Il-6, IL-8, CX3CL1 and CEBPδ (fig. S4) suggesting that 222 these compounds may be acting on multiple pathways to reduce inflammatory responses. 223 224 In mixed glial cultures, while neither digoxin or lanatoside C reduced IL-1β-induced NFκB 225 translocation in microglia (Fig. 3A to D), cytokine secretions were consistent with pericyte 226 cultures (Fig. 3E to M). This is most likely due to the low percentage of microglia present in 227 the predominantly pericyte cultures at early passage (7.261 %, ± 4.709 PU.1 positive cells). 228 This suggests that digoxin and lanatoside C do not modulate NFκB translocation-dependent 229 inflammatory effects in human microglia. 230 231 Anti-inflammatory effects of digoxin and lanatoside C in primary human brain endothelia 232

Brain endothelia lining the cerebral blood vessels are the first point of contact for therapeutic 233 drugs in the systemic circulation and express numerous active efflux transporters which 234 complicates CNS drug delivery. Additionally, brain endothelia play an important role in 235 neuroinflammatory responses generated from both the periphery and the brain parenchyma 236 by mediating recruitment, adhesion, and transcellular/paracellular immune cell trafficking 237 across the BBB through expression of cellular adhesion molecules (ICAM-1, VCAM-1) and 238 chemokines (IL-8, CCL2) (50-52). As p-glycoprotein substrates, an intact BBB would likely 239 exclude digoxin and lanatoside C entrance to parenchymal brain cells, although this barrier is 240 often weakened during inflammatory states or neurological diseases and could facilitate 241 cerebral penetrance. In order to further assess anti-inflammatory effects of digoxin and 242 lanatoside C, particularly with respect to leucocyte extravasation in the cell type most likely 243 to be exposed in vivo, inflammatory responses of primary adult human brain endothelia were 244 assessed using the aforementioned paradigm described for pericytes. 245

Expression of endothelial markers Claudin-5, ETS-related gene (ERG), zonula occludens-1 246 (ZO-1), and platelet/endothelial cell adhesion molecule-1 (PECAM/CD31) were confirmed in 247 primary cultures of human brain endothelia in vitro (Figure 4A to D). While neither digoxin 248 nor lanatoside C significantly altered basal CCL2 or ICAM-1 expression by ICC, digoxin 249 significantly increased CEBPδ expression (Fig. 4E to I). Both digoxin and lanatoside C blocked 250 IL-1β-induced secretion of all analytes measured by CBA (CCL2, sICAM-1, sVCAM-1, IL-6, IL-8, 251 CX3CL1, chemokine(C-C motif) ligand 5/regulated on activation, normal T cell expressed and 252 secreted (RANTES), granulocyte colony-stimulating factor (G-CSF), and granulocyte-253 macrophage colony-stimulating factor (GM-CSF) (Figure 3J to R). 254 255 Human meninges and choroid plexus as 3-D ex vivo targets 256

Leptomeninges envelop the brain and spinal cord and are composed of the arachnoid and pia 257 mater. The subarachnoid space within the leptomeninges is bathed in CP-derived CSF and 258 plays a crucial role in protecting the CNS. The anatomical relationship of the vascular cells, 259 meninges, and CP through the subarachnoid, Virchow-robin/perivascular space, and the CSF-260 containing ventricles allows for the exchange of fluid components be it therapeutics or less 261 desired inflammatory constituents (53). However, the meninges and CP can also contribute 262 to numerous neurological disorders, due to their intimate interactions and exposure to the 263 peripheral environment respectively through the meningeal lymphatics and the fenestrated 264 blood vessels (29, 54-56). Early immune responses occur in multiple sclerosis, and meningitis 265


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in the meninges and CP as both are sites of immune cell trafficking into the CNS (27, 57-59). 266 Further, inflammatory secretions in the subarachnoid space in the ventricular space by the CP 267 can penetrate the brain to alter cerebral functioning, likely as a consequence of glymphatic 268 influx, whereby neurovascular cells including pericytes, endothelia, and astrocytes will also 269 be subjected to cytokine presence (13, 60). In order to further investigate potential anti-270 inflammatory functions of digoxin and lanatoside C, we first established a method allowing 271 for the ex vivo cultures of human leptomeninges and CP to study inflammation, and potential 272 drug interventions in a culture system more appropriately recapitulating the in vivo 273 microenvironment than can be achieved by standard in vitro cultures. 274

Leptomeningeal explants (LME) derived from drug resistant epilepsy biopsy tissue and post-275 mortem neurologically normal and disease brains (fig. S5 A to D) were found to be viable in 276 culture, even three months after the initial isolation, as determined by live imaging using the 277 ReadyProbes Live/Dead reagents (fig. S5E to G). Immunohistochemical interrogation of cell 278 specific proteins in LME at experimental endpoints revealed mostly vascular cells, staining 279 positively for pericyte markers PDGFRβ, endothelial cells (CD31, Ulex europaeus agglutinin I 280 (UEA-1/lectin)), smooth muscle cells (αSMA), fibroblasts (PDZ and LIM domain protein 3 281 (PDLIM3)) and extracellular matrix components (collagen, type I, alpha I (COL1A1)) (Fig. 282 5A)(61). We also detected cells positive for microglia/macrophage markers Iba-1, HLA-DR and 283 CD68 in both LME and CPE after more than one month in culture (fig S6). Whilst the 284 inflammatory secretome and cytokine-specific responses of brain pericytes and endothelia 285 have been studied to some extent, significantly less is known regarding this response in the 286 leptomeninges. Secretions from LME in response LPS, IL-1β, and IFNγ closely mirrored what 287 we saw in pericytes and endothelia with adhesion molecule and chemokines responsible for 288 immune cell recruitment largely being induced by IL-1β, and LPS, and a lesser response with 289 IFNγ (22) (Fig 5B to G, fig S7A). Thus, we used cytokine profiler arrays, which allows for 290 simultaneous detection of 102 different soluble inflammatory mediators in response to the 291 aforementioned immunogenic stimuli (LPS, IL-1β, and IFNγ). As a likely reflection of their cell 292 heterogeneity, the LME demonstrated an extensive basal secretion of inflammatory 293 mediators which was differentially induced by LPS, IL-1β, and IFNγ (Fig. 5H, fig S8A). 294 Importantly, these mediators had no effect on cell viability (fig. S5F). Notably basal detection 295 of several immune mediators was observed from the LME, in particular angiogenin, 296 angiopoetin-2, chitinase 3-like, C-X-C motif chemokine 5 (CXCL5), growth/differentiation 297 factor 15 (GDF-15), insulin-like growth factor protein-2 (IGFBP-2), IL-8, osteopontin, and high 298 basal levels of serpin E1. The most drastic change in secretion in response to cytokine 299 treatment was interferon γ-induced protein-10 (IP-10/CXCL10), which is induced by both IFNγ 300 and LPS, as well as GM-CSF -by IL-1β, and IL-6 by IFNγ and IL-1β. 301 We sought to investigate whether digoxin and lanatoside C could reduce this pro-302 inflammatory response. As was observed for pericytes and endothelia, digoxin and lanatoside 303 C reduced IL-1β-induced inflammatory mediators in the leptomeninges including CCL2, 304 sICAM-1, sVCAM-1 and CX3CL1 (Fig 6A-I, fig S7B). Using the proteome profilers to interrogate 305 the extent of inhibition by digoxin or lanatoside C we found that both drugs decreased IL-1β 306 –induced secretion of G-CSF, GM-CSF, C-X-C motif chemokine 11 (CXCL11), platelet-derived 307 growth factor AA (PDGF-AA), and sVCAM-1, and they increased secretion of IL-1β dependent 308 IL-6, macrophage inflammatory protein 1-alpha (MIP1α) and TNFα consistently across three 309 LME cases (Fig 6J, fig S7B). 310


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Using the same paradigm in choroid plexus explants (CPE) we first examined the cellular 311 composition and found the highly vascularized tissue to be harbouring similar cell types to 312 the LME, with the addition of transthyretin-positive epithelia (Fig 7A and B). Similar to the 313 LME, the CPE secreted a range of cytokines in response to pro-inflammatory stimulation albeit 314 to a lesser extent than the LME (Fig 7C-L, fig S8 and S9). Just like the LME under basal 315 conditions, the CPE had undetectable levels of G-CSF, and GM-CSF, very low expression of 316 RANTES, sICAM-1, and noticeable expression of chitinase 3-like, GDF-15, IGFBP-2, IL-8, 317 osteopontin and serpin E1. Expectedly CPE cultures responded to cytokine treatment by 318 changing their secretome. Digoxin and lanatoside C pre-treatment resulted in reduced IL-1β- 319 responses such as inhibition of sICAM-1, sVCAM-1 and CX3CL1 secretion by CBA (Fig 8 A-I). 320 Proteome profilers of CPE conditioned media in contrast to LME show an increase in IL-1β 321 induced secretion of G-CSF, GM-CSF, and CXCL5, but reduced VCAM-1, angiogenin, 322 osteopontin, and interleukin 1 receptor-like 1 (ST2) by digoxin and lanatoside (Fig 8). 323

Discussion 324

Neuroinflammation is present in almost every neurological disorder and contributes to 325 disease pathogenesis by precipitating neuronal loss and BBB dysfunction (62, 63). As such, 326 the identification of therapeutic compounds that effectively attenuate neuroinflammation 327 has the potential to be beneficial in a diverse range of neurodegenerative diseases, 328 neuropsychiatric disorders, and acute brain injuries. Unfortunately, compounds that were 329 beneficial in preclinical models of neurological disease, including anti-inflammatory therapies, 330 have largely failed to effectively translate to clinical use (64, 65). Whilst the failure of anti-331 inflammatory interventions may be a function of inappropriate timing in the treatment 332 regime, the lack of translational therapeutics could be equally attributed to the frequent use 333 of rodent models in neurological drug discovery, in which responses may poorly reflect those 334 observed in humans. Failure may also be associated with targeting of general inflammatory 335 pathways irrelevant to neurodegenerative disease, suggesting that targeting distinct CNS 336 neuroinflammation pathways may prove more successful. 337

To address this issue, we utilised primary human brain pericytes to screen for anti-338 inflammatory drugs in a library consisting of >1280 FDA approved compounds. These cells 339 have the obvious benefit of being of human origin, therefore negating any species differences 340 which complicate observed responses. Additionally, unlike human microglia or astrocytes, 341 these cells are highly proliferative in vitro, allowing for efficient bulking and bio-banking of 342 these cultures to generate sufficient yields to undertake large screens (66). Due to its 343 ubiquitous presence in neurological disease, we sought to target neuroinflammation in this 344 screen in particular because together, endothelia and pericytes are the major mediators of 345 leucocyte extravasation during neurodegenerative diseases, particularly in AD and MS, 346 through CCL2-directed chemotaxis and ICAM-1 and VCAM-1-mediated adhesion (52, 67-69). 347 However, this approach would be equally suited to identify mitogenic enhancers, autophagy 348 regulators, phagocytic inducers or pro-survival drugs, utilising differing stimulation 349 paradigms. 350

The strength of the current screening approach resides in the fact that compounds underwent 351 several rounds of analysis in primary human brain cells derived from diverse healthy and 352 diseased post mortem and biopsy samples. The drug effects were consistent across different 353 donors, emphasizing the capacity of this screening platform as a tool to identify modifiers of 354


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human-specific targets taking into account the inherent human variation from the first stages 355 of drug discovery. With recent advancements in the ability to isolate and culture cells from 356 the human brain, in addition to deriving these cells from patient induced pluripotent stem 357 cells, these screening approaches can be expanded to human microglia, astrocytes, and even 358 neurons (70-72). 359

Whilst the use of non-human or immortalized cell lines has potential issues with such high-360 throughput screens, so too does the use of in vitro primary cultures themselves. Pericytes and 361 endothelia utilised here were subjected to several rounds of passaging in order to obtain 362 sufficient yields, for which phenotypic drift can occur (73, 74). It is therefore unclear whether 363 cells accurately reflect the phenotype observed in vivo. In order to analyse the anti-364 inflammatory effects of digoxin and lanatoside C in a more complex, multi-cellular model 365 without the complications of dissociated and passaged cells , a protocol to simply and 366 effectively isolate leptomeningeal and choroid plexus explant cultures was used (75). These 367 explants display excellent viability, they are easily accessible from the post-mortem brain, or 368 from neurosurgical specimens and their ease of handling makes them an attractive model. 369 The advancements in organotypic slice cultures could soon allow for coculture methods of 370 LME and CPE with human neurons to define cellular interactions in disease conditions (76). 371 The explants displayed an extensive inflammatory secretome, including cytokines, 372 chemokines, and adhesion molecules. Of equal importance, the anti-inflammatory effects 373 observed using in vitro monocultures of pericytes and endothelial cells were largely consistent 374 with our ex vivo leptomeningeal and CPE cultures, suggesting the appropriateness of both 375 model systems in identifying immune modulating compounds. The ability of these 376 compounds to attenuate inflammatory responses in several distinct cell types is promising 377 with the ability to effectively attenuate global neuroinflammation, as this response is not 378 restricted to one particular region. Furthermore, neither digoxin nor lanatoside C completely 379 blocked inflammatory responses, as can be deleterious, with a basal level of inflammatory 380 capabilities important for homeostasis and defence responses (77). 381

The utilisation of an FDA-approved compound library is advantageous as compounds have 382 already undergone safety trials in humans, easing the repurposing of drugs for other 383 disorders. As such, potential information about efficacy of such compounds can often be 384 gleaned from retrospective analysis of patient cohorts taking these compounds for other 385 indications, therefore expensive and timely clinical human safety trials are not necessary. 386 Further, extensive data are already available on their pharmacological properties (45, 72, 78). 387 Digoxin and lanatoside C are structurally very similar and well-known for their inhibitory 388 effects on the Na+/K+-ATPase pump (79). Both act similarly in cancer cell lines to inhibit 389 proliferation having actions on tumour necrosis factor-related apoptosis-inducing ligand 390 (TRAIL), Src and protein kinase C δ (PKCδ) pathways. Digoxin is approximately three times 391 more potent than lanatoside C, which is consistent with the data presented here (45, 80-82). 392 Currently, evidence for digoxin and lanatoside C in anti-inflammatory mechanisms is limited, 393 with suggested effects on leukocyte infiltration and extravasation through inhibition of 394 cytokine/chemokine secretion and reduced NFκB expression (83). Although the efficacy of 395 other cardiac glycosides as anti-inflammatory compounds in brain barrier tissues has not been 396 investigated, cardiac glycosides demonstrate a large range of chemical diversity and 397 absorption, distribution, metabolism, elimination and toxicity (ADMET) properties (79). 398 Nonetheless, we have shown that these compounds can act on multiple inflammatory 399 pathways in brain barrier cells including pericytes, endothelia, microglia, and in meningeal 400


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and choroid plexus explants. Therefore they have strong potential as anti-inflammatory drugs 401 in the context of neuroinflammation. 402

Neurodegenerative diseases are often a result of several diverging dysregulated pathways, 403 including inflammatory responses, protein aggregation, BBB breakdown, and inappropriate 404 clearance mechanisms. As such, the identification of multi-target, multi-action drugs 405 (targeting both neuroinflammatory pathways and blood-brain barrier cells) will prove more 406 effective than single target therapeutics (7). Moreover, accessibility to vascular cells via 407 perivascular spaces, and tissues such as the meninges and CP through the brain boundary 408 regions and CSF compartments should be considered in drug delivery strategies. Here we 409 demonstrate the utility of repurposed drug screens in human brain cells and identify drugs 410 for use as therapeutic agents in attacking the neurodegenerative cascade in cells and tissues 411 that can be more easily reached. Taken together, our described pipeline represents a 412 promising approach for neuroinflammatory and neurodegenerative drug development. 413

Materials and Methods 414

Study Design 415

This study was designed to identify chemical compounds that could modulate inflammatory 416 responses in human brain vascular cells. Using post-mortem brain tissue of healthy and 417 disease patients, as well as epilepsy biopsy tissue we screened 1280 FDA-approved 418 compounds against an IL-1β-induced inflammatory response. Candidates identified from 419 the initial screen by CCL2 and ICAM-1 expression in pericytes were forwarded for secretion 420 analysis, and transcription factor activity in pericytes, endothelial cells, and mixed glial 421 cultures. Meningeal and choroid plexus explants were tested as an ex vivo model of 422 neuroinflammation. 423

Tissue source 424

Biopsy human brain tissue was obtained from the Neurological Foundation Human Brain 425 Bank, with informed written consent, from the middle temporal gyrus (MTG) of patients 426 undergoing surgery for drug-resistant epilepsy. Post-mortem leptomeninges were obtained 427 from regions overlying the MTG, and CPE were derived from the lateral ventricle from one 428 hemisphere from neurologically normal individuals, or those with various neurological 429 diseases (Table S1). All brain tissue collection and processing protocols were approved by the 430 Northern Regional Ethics Committee, (AKL/88/025/AM09 New Zealand) for biopsy tissue, and 431 the University of Auckland Human Participants Ethics Committee (Ref # 011654, New Zealand) 432 for the post-mortem brain tissue. All methods were carried out in accordance with the 433 approved guidelines. 434

Primary human brain mixed glial cultures 435

Biopsy human brain tissue was obtained from the MTG of patients with drug-resistant 436 epilepsy and mixed glial cultures, containing microglia, astrocytes, and pericytes, were 437 generated as described previously (71). Mixed glial cultures were maintained in complete 438 media (DMEM/F12 with 10% FBS and 1% PSG (penicillin 100 U/mL, streptomycin 100 µg/mL, 439 L-glutamine 0.29 mg/mL)) at 37 °C with 5% CO2 until confluent. Flasks were trypsinized with 440 0.25% Trypsin- 1mM EDTA and scraped to detach firmly adherent microglia. Viable cells were 441 counted based on trypan blue exclusion and 5,000 cells/well were seeded into 96-well plates 442


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in complete media and used for experimentation after 1-3 days. All experiments performed 443 on mixed glial cultures were at passage two. 444

Primary human brain pericyte culture 445

To generate pure pericyte cultures, mixed glial cultures were sub-cultured up to passage four 446 in order to eliminate non-proliferative microglia and astrocytes as described previously (71, 447 84). Pericyte cultures were maintained in complete media at 37 °C with 5% CO2. Viable cells 448 were counted based on trypan blue exclusion and 5,000 cells/well were seeded into 96-well 449 plates in complete media and used for experimentation after 1-3 days. All experiments 450 performed on pericytes were at passages 4-9. 451

Primary human brain endothelial culture 452

Primary human endothelial cells were isolated from brain microvessels as described 453 previously (22). Endothelial cultures were maintained in Endothelia Cell Media (ECM; 454 ScienCell) at 37 °C with 5% CO2. Viable cells were counted based on trypan blue exclusion and 455 10,000 cells/well were seeded into 96-well plates in complete media and used for 456 experimentation when cells had produced a 100% confluent monolayer (typically around five 457 days). All experiments performed on endothelia were at passages three-five. 458

Compound screening 459

High-throughput screening to identify compounds which modulate inflammatory responses 460 in pericytes was performed using the Prestwick Chemical Library (Prestwick Chemical, France) 461 containing 1280 small molecules of FDA approved drugs. Controls included vehicle for 462 compounds (DMSO), TGFβ1 (1 or 10 ng/mL), vehicle for IL-1β (0.01% BSA in PBS), or media 463 alone. Pericytes were pre-treated with compounds (or controls) in duplicate at 10 µM for 24 464 hours before treatment with IL-1β (0.05 ng/mL) for another 24 hours. At endpoint cells were 465 fixed and immunostained for CCL2 and ICAM-1 and nuclei were counterstained with Hoechst 466 33258 (as detailed below). Cells were imaged using the ImageXpress® Micro XLS automated 467 fluorescent microscope and total Hoechst positive cell counts and the integrated intensity per 468 cell of CCL2 and ICAM-1 was quantified as described previously (17). 469

Immunocytochemistry and high-throughput image analysis 470

At endpoint cells were fixed in 4% paraformaldehyde for 15 minutes and 471 washed/permeabilized with phosphate buffered saline (PBS) with 0.2% Triton X-100™ (PBS-472 T). Cells were incubated with primary antibodies diluted in goat immunobuffer (1% goat or 473 donkey serum, 0.2% Triton X-100™, and 0.04% thiomersal in PBS) overnight at 4 oC, (dilutions 474 of antibodies are listed in Table S2). Cells were washed again in PBS-T and incubated with 475 fluorescently conjugated secondary antibodies for two hours at room temperature. Nuclei 476 were counterstained by a 15 minute incubation with 20 μM Hoechst 33258 (Sigma). 477 Quantitative analysis of intensity measures and scoring of positively stained cells was 478 performed using the Cell Scoring, Multiwavelength Cell Scoring, and Nuclear Translocation 479 modules on MetaXpress® software (Molecular Devices) as previously described (48) . 480

Cytometric bead array 481


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Conditioned media was collected from samples and centrifuged at 160 x g for five minutes to 482 collect possible cells and debris. The supernatant was obtained and stored at 20 °C. Analyte 483 concentrations were measured by cytometric bead array (CBA; BD Biosciences, CA, USA) as 484 described previously (85). CBA samples were run on an Accuri C6 flow-cytometer (BD 485 Biosciences, CA, USA). Data were analysed using FCAP-array software (version 3.1) (BD 486 Biosciences, CA, USA) to convert fluorescent intensity values to concentrations utilising a 11-487 point standard curve (0-10,000 pg/mL). Concentrations were normalised to total cells as 488 determined by Hoechst positive counts for monolayer cells. 489 490 Human NFκB pathway array kit 491 Human pericytes were seeded into 10 cm dishes and allowed to reach confluency. Pericytes 492 were treated with DMSO (0.01%) or lanatoside C (0.1 µM) for 24 hours then treated with IL-493 1β (0.05 ng/mL) for 30 minutes. Cells were lysed on ice with kit lysis buffer containing 494 protease inhibitor tablets (Roche), scraped into pre-chilled tubes and incubated on ice for 30 495 minutes with intermittent vortexing. Lysates were centrifuged at 14,000 x g for 15 minutes, 496 and protein concentration of the supernatant was quantified using the DC protein 497 quantification kit (Bio-Rad). Samples were stored at -80 oC until array analysis. Arrays were 498 carried out following the manufacturer’s instructions, with 500 µg of protein from each 499 sample used for analysis. Membranes were imaged using the LI-COR Odyssey® FC imaging 500 system (LI-COR Biosciences). Intensity of individual spots was determined using Image 501 Studio™ Lite and values were normalized to the average of the six reference spots on each 502 blot. 503 504 Cytokine profiler 505 506 Conditioned media from 3-6 leptomeningeal explants was pooled, centrifuged at 160 x g for 507 five minutes, and the supernatant was collected and stored at -20 °C. Secretome analysis of 508 the clarified media was performed using the Proteome Profiler™ Human XL Cytokine Array Kit 509 (R & D Systems), as per manufacturer’s instructions. Chemiluminescent detection of the 510 membranes was performed on the LI-COR Odyssey® FC (LI-COR Biosciences). Intensity of 511 individual spots was determined using Image Studio™ Lite and values were normalized to the 512 average of the six reference spots on each blot. 513 514 EdU proliferation assay 515

Proliferation of pericytes was determined by incorporation of the thymine analogue 5-516 ethynyl-2'-deoxyuridine (EdU) with the Click-iT®Assay Kit (Life Technologies C10340) 517 according to manufacturer’s instructions. Briefly, EdU (5 µM) was added to cells 24 hours prior 518 to endpoint and incubated at 37 oC. Cells were fixed with 4% PFA for 15 minutes at room 519 temperature, rinsed with 3% BSA in PBS, and permeabilized with 0.5% Triton X-100 in PBS for 520 20 minutes at room temperature. Cells were washed twice with 3% BSA in PBS and then EdU 521 reaction cocktail was added for 30 minutes at room temperature protected from light. Cells 522 were then washed once more with 3% BSA in PBS and imaged as described above. 523

Primary human leptomeningeal and choroid plexus explant culture 524

Leptomeninges were removed by gross dissection from the brain overlying the MTG, from 525 autopsy tissue of neurologically normal or pathologically confirmed cases of 526


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neurodegenerative disorders (AD, PD, HD, and FTD), as previously described (86). 527 Leptomeningeal tissue was washed in complete media in sterile petri dishes and dissected 528 into pieces ~ 2 mm3. Leptomeningeal explants were placed in individual wells in 500 μL of 529 complete media in a 24-well plate allowing them to remain in suspension. Media changes 530 were performed twice a week. Explants that failed to alter media acidification, as determined 531 by a phenol red colour change, were deemed non-viable and discarded. Explants were 532 cultured for at least a week before using for experiments to allow equilibration but remained 533 viable for up to four months after initial isolation. For functional studies, individual explants 534 were placed in 100 μL of media within a 96-well plate. 535

Explant viability and histological processing 536

Cell death was determined using the ReadyProbes™ cell viability imaging kit (Invitrogen). 537 NucBlue live reagent and NucGreen dead reagent stains were diluted into media (2 drops per 538 mL blue, 1 drop per mL green), and added to cells in culture 30 minutes prior to endpoint and 539 incubated at 37 oC for 30 minutes. Z-stacks (200-250 slices, 5 μm step) of explants were then 540 acquired using the ImageXpress high content imaging system (Molecular Devices). 2-D 541 projections were used for analysis of positively labelled nuclei with the Custom Module Editor 542 (image analysis pipeline described in detail in supplementary data (fig. S5). 543

Immunohistochemical staining on formalin fixed, paraffin embedded explants, sectioned at 544 7µm was done as previously described (87). Antigen retrieval with TRIS-EDTA (pH 9) was 545 performed, followed by blocking with 10% donkey serum prior to antibody incubation. 546 Sections were incubated with primary antibodies (for dilutions see Table S2) overnight at 4 547 oC, rinsed in PBS then incubated with secondary antibodies and Hoechst 33258 (SIGMA) for 3 548 hours at room temperature in the dark. Sections were imaged using the 20X objective on the 549 Metasystems V-slide scanning microscope and stitched to generate images of whole explants. 550 Control sections where the primary antibody was omitted showed no immunoreactivity. The 551 control experiments showed that the secondary antibodies did not cross-react with each 552 other. All confocal recordings were done using an FV1000 confocal microscope (Olympus) 553 with a 40X oil immersion lens (NA 1.00). 554

Statistical Analysis 555

All cell culture experiments were performed three separate times with cells from different 556 donors. Data were normalized to vehicle controls were indicated in figure legends. Statistical 557 test were performed using Graphpad Prism software, one-way, or two-way ANOVA with 558 Tukey’s post hoc analysis. Data are presented as the mean ± SEM. 559

Supplementary Materials 560

Fig. S1. Controls from compound screen and follow-up screen. 561 Fig. S2. NFκB pathway profiler original blots. 562 Fig. S3. Concentration response curves for cytokine secretion and viability from pericytes 563 treated with digoxin or lanatoside C (raw data from three cases). 564 Fig. S4. Gene expression data from pericytes treated with IL-1β and digoxin or lanatoside C. 565 Fig. S5. Using explant cultures derived from human meninges and choroid plexus for drug 566 testing. 567 Fig. S6. Microglia/macrophage markers in leptomeningeal and choroid plexus explants. 568


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Fig. S7. Raw data from CBA analysis of leptomeningeal explant secretions. 569 Fig. S8. Human cytokine proteome profiler original blots and select hits comparison. 570 Fig. S9. Raw data from CBA analysis choroid plexus secretions 571

572

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82. M.-W. Chao, T.-H. Chen, H.-L. Huang, Y.-W. Chang, W.-C. HuangFu, Y.-C. Lee, C.-M. 817 Teng, S.-L. Pan, Lanatoside C, a cardiac glycoside, acts through protein kinase Cδ to 818 cause apoptosis of human hepatocellular carcinoma cells. Scientific Reports 7, (2017). 819

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835

Acknowledgments 836 We would like to thank the donors for their generous gift of brain tissue for research. We also 837 thank staff at Auckland Hospital and Marika Eszes (Technical Officer at the Neurological 838 Foundation Human Brain Bank). This work was supported by a Programme Grant from the 839 Health Research Council of New Zealand, the Sir Thomas and Lady Duncan Trust, the Coker 840 Charitable Trust and the Hugh Green Foundation. 841 842 Conflict of interest 843 The authors declare no conflict of interest. 844 845


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Figures 846 847

848 849 Fig. 1: High throughput screening of pericytes identifies inflammatory-modulating compounds. 850 (A-B) Pericytes were treated with IL-1β for 24 hours and immunostained for CCL2 and ICAM-1 protein 851 expression. Integrated intensity per cell was calculated using total cells (Hoechst), n = 3, significance 852 determined using one-way ANOVA with Dunnett’s correction for multiple comparisons, 853 ****p<0.0001. (C-D) Pericytes were screened for compounds that can modify IL-1β-induced CCL2 or 854 ICAM-1 expression using the Prestwick chemical library. Integrated intensity per cell was calculated 855 using total cell counts and normalized to IL-1β + vehicle condition (arrow)( blue arrow-digoxin, red 856 arrow-lanatoside C, black arrows-hit compounds tested in (G)). Internal controls were present on each 857 drug plate (TGFβ1, 1 or 10 ng/mL). E-F). Hits identified using cut-off criteria from primary screen that 858 modified CCL2 or ICAM-1 expression with known therapeutic uses. (G) Conditioned media from 859 pericytes pre-treated with hit compounds for 24 hours, then stimulated with IL-1β (0.05 ng/mL) for 24 860 hours was analysed by CBA, data was normalized to cell number and presented as the logged value of 861 cytokine secretion in pg/mL/10,000 cells. Control (vehicle for IL-1β (0.01% BSA in PBS), IL-1β (0.05 862 ng/mL + dimethylsulfoxide (DMSO))(n = 2, media pooled from triplicate wells for analysis). (H) Drug 863 screening pipeline in primary human brain pericytes. Initial screen in pericytes completed in duplicate 864 wells and secondary screen in 83 compounds that met cut-off criteria was completed in triplicate 865


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wells. Effects that were reproduced were examined using a 3 point concentration test, then tested for 866 effects on cell secretion (data in fig. S1). 867 868

869 Fig. 2: Cardiac glycosides digoxin and lanatoside C modify inflammation-dependent transcriptional 870 responses in pericytes. 871 (A-L) Conditioned media from pericytes after 24 hours of vehicle, digoxin or lanatoside C (125nM 872 points selected from concentration curves in fig. S2) pre-treatment prior to stimulation with IL1β (0.05 873 ng/mL) for 24 hours (n = 3). (M-P) Pericytes pre-treated with digoxin or lanatoside C for 24 hours were 874 treated with IL-1β (0.05 ng/mL) for 1 hour, and stained for CEBPδ or NFκB, (representative images (M 875 and O, scale = 100 µm), (N) quantification of CEBPδ percent positive cells, and (P) percent NFκB nuclear 876 translocation were compared to control conditions. Two-way ANOVA with Tukey’s multiple 877 comparison test, ***p<0.001, **p<0.01, *p<0.05, (n = 3). (Q) Pericytes were pre-treated with vehicle 878 or lanatoside C (1 µM) for 24 hours, then treated with vehicle or IL-1β (0.05 ng/mL) for 30 minutes. 879 Lysates (500 µg per sample) were analysed using the human NFκB array kit. Average intensity values 880 from duplicate spots were normalised to the average intensity of the reference spots on each blot. R) 881 Select targets from NFκB profiler presented as fold change in intensity from vehicle. 882 883


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884 Fig. 3: Cardiac glycosides modulate cytokine secretions from mixed glial cultures of microglia and 885 pericytes. 886 (A, B) Mixed glial cultures were treated with cardiac glycosides for 24 hours prior to stimulation with 887 IL1β for 1 hour (A representative images). Inflammatory response was assessed by NFκB nuclear 888 translocation in PU.1 positive microglia (C, D). (E-M) Conditioned media from mixed glial cultures after 889 24 hours of vehicle, digoxin (100 nM) or lanatoside C (1 µM) pretreatment prior to stimulation with 890 IL1β (0.05 ng/mL) for 24 hours (n = 3). Two-way ANOVA with Tukey’s multiple comparison test, 891 ***p<0.001, **p<0.01, *p<0.05. 892 893


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894


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Fig. 4: Inflammatory cytokine secretion is blocked by both digoxin and lanatoside C in primary human 895 brain endothelial cells. 896 Endothelial cells derived from human brain tissue express endothelial markers, Claudin-5 (A), ZO-1, 897 (B), ERG (C), and CD31 (D). (E-G) Human endothelial cells were pretreated with digoxin (100 nM) or 898 lanatoside C (1 µM) for 24 hours, followed by 24 hours of IL-1β (0.05 ng/mL) treatment. Staining 899 quantification of CCL2 (E), ICAM-1 (F), and nuclear CEBPδ (G), in endothelial cells. Representative 900 images of (n = 3) (H, I). (J-R) Conditioned media was analysed by CBA in endothelial cells treated as 901 above (n = 3). Cytokine/chemokine concentration was normalized to total cell counts (Hoechst)). Two-902 way ANOVA with Tukey’s multiple comparison test ***p<0.001, **p<0.01, *p<0.05. 903 904

905 Figure 5: Characterization of leptomeningeal explant cell types and inflammatory signature. 906 A) Immunohistochemical staining of LME for vascular cells types including pericytes (PDGFRβ) 907 endothelial cells (CD31), fibroblasts (PDLIM3), smooth muscle cells (αSMA) and extracellular matrix 908 protein deposition (COL1A1). (B-G) Secretions from meningeal explants following inflammatory 909 stimulation were investigated using cytometric bead arrays (n = 2 cases, 3-4 explants per case), values 910 normalized to vehicle condition, raw values Fig S7, 8. (H) Proteome profilers were used to detect 911 secretions from human brain meningeal explants (pooled from 5 explants per condition, one case) 912


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after 24 hours of inflammatory stimulation with vehicle, IFNγ, IL-1β, or LPS (10 ng/mL), raw intensities 913 were normalized to reference spots. 914 915

916 Figure 6. Cardiac glycosides modulate inflammatory responses in leptomeningeal explant 917 cultures. 918 A-I) meningeal explant secretions were measured using CBA (n = 3-5 explants per condition, 919 5 cases). Secretions in pg/mL were normalized to vehicle for each case-arbitrary units (A.U.) 920 except for G-CSF, GM-CSF and RANTES where cytokines were below the detection level in 921 vehicle conditions. Two-way ANOVA with Tukey’s multiple comparison test ****p<0.0001, 922 ***p< 0.001, **p< 0.01, *p<0.05. J) Proteome profilers were used to characterise LME 923 secretions in response to vehicle or IL-1β (10 ng/mL) with either digoxin or lanatoside C pre-924


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treatment (10 µM). Average intensity values from proteome profilers were normalised to 925 the reference spots on each blot (heatmap is average of n=3 cases, each pooled from 3-4 926 explants per case). 927 928

929 Figure 7: Characterization of choroid plexus explant cell types and inflammatory signature. 930 (A, B) Immunohistochemical staining of CPE for vascular cells types including pericytes (PDGFRβ) 931 endothelial cells (CD31), fibroblasts (PDLIM3), smooth muscle cells (αSMA) and epithelial cells 932 (transthyretin (TTR)). (C-K) Secretions from CPE following inflammatory stimulation were 933 investigated using cytometric bead arrays (n = 3 cases, 3 explants per case), values normalized to 934 vehicle condition, raw values Fig S8, 9. One way ANOVA, Dunnett’s multiple comparisons 935 test, ***p< 0.001, **p< 0.01, *p<0.05. (L) Proteome profilers were used to detect secretions from 936 human brain choroid plexus explants (average of n = 2 cases each pooled from 3 explants per 937 condition) after 24 hours of inflammatory stimulation with vehicle, IFNγ, IL-1β, or LPS (10 ng/mL), 938 raw intensities were normalized to reference spots. 939 940


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941 Figure 8. Cardiac glycosides modulate inflammatory responses in choroid plexus explant 942 cultures. 943 A-I) Choroid plexus explants were treated with vehicle, digoxin or lanatoside C (both 10 µM) 944 for 24 hours followed by IL1β (10 ng/mL) for 24 hours. Secretions were measured using CBA 945 (n=2 cases, 3 explants per case). Values normalized to vehicle for each case, except for G-946 CSF, GM-CSF and RANTES as vehicle condition was under the level of detection, (raw data 947 provided in supplementary, Fig S9). J) Proteome profiler analysis of pooled choroid plexus 948


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explant secretions (3 pooled explants per case). Data are presented from the average of 949 each case normalised to reference point. 950 951 Table S1. Human tissue sources used in this study. 952

Case Tissue source Confirmed diagnosis Age Sex PMD (hr)

Experiment

E203 MTG Temporal lobe epilepsy 46 F <1 ML E204 MTG Temporal lobe epilepsy 45 F <1 ML E206 MTG Temporal lobe epilepsy 29 F <1 ML E208 MTG Temporal lobe epilepsy 52 F <1 ML E211 MTG Temporal lobe epilepsy 51 F <1 ML E213 MTG Temporal lobe epilepsy 23 M <1 ML E214 MTG Temporal lobe epilepsy 35 F <1 ML E215 MTG Temporal lobe epilepsy 29 F <1 ML E216 MTG Temporal lobe epilepsy 38 M <1 ML SS37 TL Paediatric epilepsy 9 M <1 ML SS42 TL Paediatric epilepsy 8 M <1 ML SS48 TL Paediatric epilepsy 10 F <1 ML SS50 TL Paediatric epilepsy 3 F <1 ML SS54 TL Paediatric epilepsy 25w M <1 ML H238 MTG, meninges normal 63 F 16 ML, LME H239 MTG, meninges normal 64 M 15.5 ML, LME H250 meninges normal 93 F 19 LME H253 meninges normal 96 M 24 LME, CPE FTD6 Meninges, CP Frontotemporal dementia 76 M 7 LME, CPE HC166 Meninges, CP Huntington’s disease 84 F 22.5 LME, CPE HC171 Meninges, CP Huntington’s disease 51 M 24 LME, CPE PD85 meninges/CP Parkinson’s disease 73 M 4 LME, CPE AZ129 meninges Alzheimer’s disease 81 M 4.5 LME AZ133 meninges/CP Alzheimer’s disease 96 M 21 LME, CPE

MTG-middle temporal gyrus, ML-monolayer cultures, CP-choroid plexus, LME-leptomeningeal 953 explants, CPE-choroid plexus explants 954 955 Table S2. Antibodies and stains used in this study. 956

Antibody (species) Catalogue Number (Source) Dilution αSMA (mouse) IS611 (Dako) 1/100 IHC CCL2 (MCP-1) (Rabbit) ab-9669 (Abcam) 1/500 ICC CD31 (mouse) M0823 (Dako) 1/500 ICC, 1/100 IHC CD68 (KP1)(mouse) Ab-955(Abcam) 1/500 IHC CEBPδ (mouse) sc-365546 (Santa Cruz) 1/500 ICC Claudin-5 (rabbit) ab131259 (Abcam) 1/5000 ICC COL1A1 (mouse) sc-293182 (Santa Cruz) 1/50 IHC HLA-DR (mouse) M0775 (Dako) 1/1000 IHC IBA1 (goat) ab-5076 (Abcam) 1/500 IHC ICAM-1 (mouse) sc-107 (Santa Cruz) 1/500 ICC IL-6 (goat) AF-206 (R&D Systems) 1/2000 ICC KCNJ8/Kir6.1 (rabbit) PA5-56628 1/100 IHC NFκBp65 (rabbit) sc-107 (Santa Cruz) 1/500 ICC PDGFRβ (goat) AF-385 (R&D Systems) 1/2000 ICC


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1/200 IHC PDGFRβ (rabbit) 3169 (Cell Signalling) 1/100 IHC PDLIM3 (rabbit) PA5-52099 (Thermo Fisher Scientific) 1/50 IHC PU.1 (rabbit) 2258 (Cell Signalling) 1/500 ICC SMAD2/3 (mouse) sc-133098 (Santa Cruz) 1/500 ICC STAT1 (rabbit) 14994 (Cell Signalling) 1/500 ICC Streptavidin Alexa Fluor 647 S21374 (Invitrogen) 2 mg/mL (1/500) Transthyretin /Pre-albumin (rabbit)

A0002 (Dako) 1/1000

ZO-1 (mouse) 339100 (Invitrogen) 1/500 ICC Lectin (UEA-1) L8262 (SIGMA) 1/1000 IHC Hoechst 33258 SIGMA 1/500 ICC

1/10000 IHC IHC-immunohistochemistry, ICC-immunocytochemistry 957


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